Riboswitches are structured RNAs that undergo significant conformational changes in response to specific ligands as a mechanism to regulate gene expression. In addition to several proteins, two different classes of c-di-GMP riboswitches have been implicated in the cyclic diguanosine monophosphate (c-di-GMP) second-messenger pathway, a ubiquitous signaling method in bacteria. The crystal structures for both classes of c-di-GMP riboswitches have been solved showing two different mechanisms for c-di-GMP recognition. These riboswitches are located in the 5'-untranslated regions of genes involved in biofilm formation, cell motility, and virulence factors. Since c-di-GMP riboswitches are prevalent in bacterial species responsible for tetanus, anthrax, botulism, and cholera among others, their characterization has direct therapeutic applications. Although the structure of both classes of riboswitches has been extensively characterized, very few experiments have been performed in vivo. The Strobel lab has synthesized a series of c-di-GMP analogues to differentially study the ligand binding characteristics in both c-di-GMP riboswitches. Several of these ligands also show resistance to intracellular cleavage and are ideal for studying the behavior of c-di-GMP riboswitches in the cell. This proposal seeks to determine the functions of c-di-GMP in the cell and define the role of the c-di-GMP riboswitch on bacterial gene expression using a multidisciplinary approach spanning biochemistry, structural biology, microbiology, and genetics.